It has been known that polymorphisms of the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene (CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910)), polymorphism of CYP4F2 (cytochrome P450, family 4, subfamily F, polypeptide 2) gene (rs2108622) and polymorphisms of VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene (VKORC1-1639 (rs9923231) of or VKORC1 1173 (rs9934438)) significantly impact the maintenance dose for the drug warfarin (Clinical Chemistry 55:4, 804-812 (2009); Blood. 2008 Apr. 15; 111(8):4106-12. Epub 2008 Feb. 4.; Ann Clin Lab Sci. 2011 Summer; 41(3):229-35). Before prescribing the drug, prescreening patients for their genotypes will facilitate a faster individualized determination of the proper maintenance dose, minimizing the risk of adverse reactions and reoccurrence of thromboembolic episodes. However, if genotyping is performed to determine the loading therapy can be delayed by several hours to 1 day.
Loop-mediated isothermal amplification (LAMP), a special type of nucleic acid amplification, is a new technique developed by Eiken Genome of Japan in 2000. This method is able to complete an amplification of DNA or RNA in one hour by using four primers in an environment of 60-65° C. Since the loop-mediated isothermal amplification has the advantages of speed, simplicity of isothermal amplification, and assay-design flexibility, the loop-mediated isothermal amplification is very suitable for various types of nucleic acid testing, such as genotype identification.
FIGS. 1A and 1B illustrate the basic principles of the loop-mediated isothermal amplification, and the detailed principles and operation instructions of this method can be referred to in U.S. Pat. No. 7,175,985 B1 and the animation illustration provided on the website of Eiken Genome of Japan.
According to FIGS. 1A and 1B, it is understood that for the loop-mediated isothermal amplification, six regions are selected in a target gene 100, which are F1, F2, F3, B1c, B2c and B3c, respectively, and F1c, F2c, F3c, B1, B2 and B3 in the other strand shown in FIGS. 1A and 1B are complementary to the F1c, F2c, F3c, B1, B2 and B3 regions mentioned above, respectively. In this method, at least one of F1 and B1c regions which are selected has to contain a mutation or single-nucleotide polymorphism site which is targeted for detection, and the enzyme which is adopted is Bst DNA polymerase which is capable of performing amplification reaction in isothermality and opening the double-strand structure of a target or template DNA during amplification.
Furthermore, in this method, four primers are adopted, and they are forward inner primer (FIP) 101, forward outer primer 103, backward inner primer (BIP) 105 and backward outer primer 107.
The sequence of the forward inner primer (FIP) 101 consists of a first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of F1c region which is predicated) and a second segment (the sequence of the F2 region). For example, if the F1 region of the target gene is predicated as a sequence from a wild type, the sequence of the first segment is the complementary strand of the F1 region from the wild type, in contrast, if the F1 region of the target gene is predicated as a sequence from a mutant type, the sequence of the first segment is the complementary strand of the F1 region from the mutant type.
The sequence of the forward outer primer 103 is the sequence of the F3 region
Moreover, the sequence of the backward inner primer (BIP) 105 consists of a third segment (the sequence of the B1c region which is predicated, that is the complementary strand of the sequence of the B1 region which is predicated) and a fourth segment (the complementary strand of the sequence of the B2c region, that is the sequence of the B2 region). For example, if the B1c region of the target gene is predicated as sequence from a wild type, the sequence of the third segment is the sequence of the B1c region from the wild type, in contrast, if the B1c region of the target gene is predicated as a sequence from a mutant type, the sequence of the third segment is the sequence of the B1c region from the mutant type.
Furthermore, the sequence of the backward outer primer 107 is the complementary strand of the sequence of the B3c region (namely, the sequence of the B3 region)
While performing the loop-mediated isothermal amplification, the second segment (the sequence of the F2 region) of the forward inner primer (FIP) 101 will anneal to the F2c region of the other strand of the target gene mentioned above and proceed to a complementary strand synthesis reaction, and a first strand which has the sequences of the first segment (the complementary strand of the sequence of the F1 region which is predicated, that is the sequence of the F1c region which is predicated), second segment (the sequence of the B2 region), F1, B1c, B2c and B3 regions is synthesized, and the forward outer primer 103 will push the first strand aside and thus a second strand which has the sequences of F3, F2, F1, B1c, B2c and B3 regions is synthesized.
Next, the fourth segment (the sequence of the B2 region) of the backward inner primer (BIP) 105 anneals to the B2c region of the foregoing first strand. And a third strand which has the sequence of the B1c region as predicated, the sequences of B2, B1, F1c, F2c, and the sequence of F1 which is predicated is synthesized by using the first strand as a template.
After that, the backward outer primer 107 will push the third strand aside and thus a fourth strand which has the sequences of the B3, B2, B1, F1c and F2c regions and the sequence of the F1 region which is predicated is synthesized. The predicated B1c region of the third strand and B1 region of the third strand will result in self-annealing, and similarly the predicated F1 region of the third strand and F1c region of the third strand will also result in self-annealing. Thus the third strand will become a strand with two ends each have a loop formed.
Then, the forward inner primer (FIP) and backward inner primer (BIP) continue the complementary strand synthesis reaction by using the complementary strand synthesis products of the third strand and/or the complementary strand thereof as the template in turn, and a double-strand product which has a plurality of loops is formed (please refer to FIG. 1B).
In addition, FIG. 2A shows results obtained from performing the loop-mediated isothermal amplification, the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated being the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively. When the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, since the synthesized third strand which is mentioned in the foregoing paragraph and which has the sequence of the B1c region which is predicated, the sequences of B2, B1, F1c, F2c, and the sequence of the F1 which is predicated, is able to become a strand whose two ends each have a loop formed, the complementary strand synthesis reaction can continue.
In contrast, FIG. 2B shows results obtained from performing the loop-mediated isothermal amplification The sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated is not the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively. When the sequence of the F1 region which is predicated and the sequence of the B1c region which is predicated are not the same as the sequence of the F1 region and the sequence of the B1c region of the target gene, respectively, the third strand can not self-anneal to form a strand whose two ends each have a loop formed. Thus complementary strand synthesis can not be continued.
Therefore, according to that mentioned above, by using appropriate primers for wild type and mutant type, respectively, a mutation and/or polymorphism of a specific region in a target nucleotide sequence can be detected.
However, for rs1799853 of the CYP2C9*2 gene, rs1057910 of the CYP2C9*3 gene, rs2108622 of the CYP4F2 gene, rs9923231 of VKORC1-1639 and rs9934438 of VKORC1 1173, primers designed by the software, PrimerExplorer V4, which is provided by Eiken Genome on its website to perform the loop-mediated isothermal amplification, no amplification results can be detected for previous mentioned five genes.
Therefore, a new kit is needed which can be used to quickly and accurately detect the single-nucleotide polymorphism of CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910) of the CYP2C9 gene, rs2108622 of CYP4F2 gene, VKORC1-1639 (rs9923231) and VKORC1 1173 (rs9934438) of VKORC1 gene.